Co-differentiation of monocytes from allogeneic donors

ABSTRACT

Disclosed is a method of producing non-exhausted immature dendritic cells (DCs) originating from at two different, allogeneic donors. In the method, a mixture of allogeneic leukocytes, which allogeneic leukocytes have been obtained from at least two different, allogeneic donors is provided. Subsequently, allogeneic monocytes are isolated from the mixture of allogeneic leukocytes. Thereafter, non-exhausted immature DCs are generated from said isolated allogeneic monocytes.

FIELD OF THE INVENTION

The present invention relates to a method for producing non-exhaustedimmature monocyte-derived human dendritic cells (DC).

BACKGROUND

Traditional cancer therapies, such as surgery, radiation, andchemotherapy, are often insufficient in treating patients and usuallycause severe side effects.

Immunotherapy has shown promise as an alternative treatment method withless negative side effects.

It is now well established that the immune system has cells,particularly CD8+ cytotoxic T lymphocytes (CTLs), which can recognizeand potentially kill tumor cells. Nevertheless, a major problem is thatthe killing ability of these T cells are either not induced or onlyweakly induced in cancer patients. One possibility is that there isinadequate tumor antigen presentation and co-stimulation by dendriticcells (DCs), “nature's adjuvant” for eliciting a functional andtumor-specific T cell immunity in cancer patients.

Existing cancer immunotherapy strategies mainly focus on antigen-loadedautologous, patient-derived DCs, which have been differentiated andantigen-loaded ex vivo. The underlying premise of this approach is thatthe efficiency and control provided by ex vivo manipulation of the DCsgenerates DCs with strong antigen-presenting and co-stimulatorycapacity. The quality of the T cell response depends on the ability ofthese autologous DCs to present tumor antigens to T cells in aMHC-restricted manner (DCs and T cells have to be from the sameindividual) in draining lymph nodes and thus create a tumor-specific Tcell response.

Monocyte-derived, autologous DCs are the most commonly used DCs in pilotstudies, as it is possible to obtain billions of monocytes fromperipheral blood leukocytes collected by leukapheresis, a laborious andtime-consuming procedure in which white blood cells are separated fromcirculating blood. Several methods are available to subsequently enrichmonocytes and two of these methods, elutriation and antibody/beadisolation, can also be performed in conformity with Good ManufacturingPractice (GMP) guidelines.

The monocytes are subsequently cultivated in media supplemented withGM-CSF and IL-4 for 4-7 days, leading to their differentiation intoimmature DCs, which immature DCs are characterized by their outstandingcapacity to produce large amounts of pro-inflammatory chemokines andcytokines upon subsequent stimulation with certain types of activatingfactors (Sallusto et al, Eur J Immunol, 1999. 29:1617; Napolitani et al,Natuer Immunology 2005. 6:769). The stimulated DCs are usuallypre-pulsed with relevant tumor antigen(s) and activated for 1-2 daysbefore vaccination. However, the immune responses to such DC-basedvaccines are often weak, and clinical responses are rarely complete andlong lasting.

Little has been known regarding the fate and function of ex vivogenerated autologous DCs after they have been injected. In the humansetting, the migration pattern of injected vaccine DCs was recentlytracked in vivo and notably, less than 5% of the injected DCs reachedthe draining lymph nodes while the majority of DCs remained at theinjection site. These locally trapped vaccine DCs rapidly lost theirviability and were subsequently cleared by recruited antigen-presentingcells.

Data has now been provided that injected vaccine DCs that have beenactivated ex vivo during a limited time-period (i.e. 6 to 18 h) becomepro-inflammatory (PI) DCs, which are able to indirectly prime nativeCD8+ T cells in vivo by acting as a pure local immune adjuvant. Thisadjuvant function of injected PI-DCs is strictly dependent on theirongoing secretion of certain DC and NK-cell recruiting chemokines at thetime of administration (after removal of activating factors). SuchPI-DCs also express/secrete factors that induce activation of recruitedendogenous NK-cells and DCs at the vaccination site. In contrast toPI-DCs, long-time (i.e. >24 h) activated DCs, which have been commonlyused in clinical trial, are characterized by their “exhausted” state(Langenkamp et al 2000), and therefore unable to secrete desirablechemokines and DC-activating factors at the time of administration.

In conclusion, PI-DCs not only can act as direct stimulators ofMHC-compatible autologous T cells but also act as an adjuvant producinglarge quantities of pro-inflammatory chemokines and cytokines at thetime of administration. Local injection of PI-DCs will lead torecruitment and activation of other immune cells, including circulatingNK cells and DC-precursors. If the injected PI-DCs have been preloadedwith relevant tumor antigens or injected directly into an existing tumorlesion, recruited endogenous DCs will engulf dying vaccine cellsexpressing relevant tumor antigens or dying antigen-expressing tumorcells, respectively. After activation these recruited and subsequentlyantigen-loaded endogenous DCs will migrate to draining lymph nodes werethey prime tumor-specific T cells in a MHC-restricted manner (Liu et al,2008). This conclusion is supported by data from several recentpre-clinical studies in which tumor growth was significantly reduced bytherapeutic vaccinations with non-exhausted MHC-incompatible, allogeneicPI-DCs (Alder et al 2008, Siders et al 2009, Edlich et al 2010)

The strong adjuvant function by PI-DCs, which importantly don't requireMHC-compatibility between PI-DCs and patient T cells, thereforeintroduces the possibility of using pre-produced and freeze-storedMHC-incompatible, allogeneic, PI-DCs as “off the shelf” vaccines,representing a viable, practical alternative to the current custom-made,patient-specific DC vaccines. The use of such MHC-incompatible,allogeneic, PI-DCs is disclosed in EP 1 509 244 B1 and WO 2011/098516.

For ethical reasons, large scale procurement of monocytes from normalblood donors by leukapheresis for the sole purpose of commerciallarge-scale vaccine production for clinical use is not feasible. Inpractice, the available raw material, i.e. monocytes, for PI-DCproduction is therefore restricted to monocytes obtained fromwaste-product (buffy coats and/or used leukocyte depeltion filteers) inthe course of separating unwanted leukocytes from different whole bloodcomponents or monocytes obtained from buffy coats at blood banks.

However, the total number of monocytes which can be isolated from eachbuffy coat or from each blood bag-leukocyte depletion filter is usuallyless than 200 millions (Ebner S et al., Generation of large numbers ofhuman dendritic cells from whole blood passaged through leukocyteremoval filters: an alternative to standard buffy coats J ImmunolMethods 252 (2001), leading to unacceptable high costs for enrichmentand subsequent DC-differentiation of separate batches of monocytes (eachbatch derived from one single donor) with methods that are in conformitywith Good Manufacturing Practice (GMP) guidelines according to the art.

Thus, there is a need in the art for a method for large scale andcost-effective clinical grade production of non-exhausted immaturedendritic cells from monocyte-containing waste-product derived fromblood banks.

SUMMARY

Consequently, the present invention seeks to mitigate, alleviate,eliminate or circumvent one or more of the above-identified potentialdeficiencies in the art and disadvantages singly or in any combinationby providing a method a method of producing non-exhausted immaturedendritic cells (DCs) originating from at least two different,allogeneic donors. In the method, a mixture of allogeneic leukocytes,which allogeneic leukocytes have been obtained from at least twodifferent, allogeneic donors is provided. Subsequently, allogeneicmonocytes are isolated from the mixture of allogeneic leukocytes toprovide monocyte-enriched allogeneic leukocytes. Thereafter,non-exhausted immature DCs are generated from the monocyte-enrichedallogeneic leukocytes, by co-culturing the monocyte-enriched allogeneicleukocytes for 2 to 7 days in aqueous cell culture medium free fromnon-human serum. The medium is supplemented with interleukin-4 (IL-4)and granulocyte-macrophage colony stimulating factor (GM-CSF).

In contrast to the prevailing prejudice, the generated monocyte-derivedimmature dendritic cells are non-exhausted and thus able to producesubstantial amounts of pro-inflammatory chemokines and pro-inflammatorycytokines in a sustained fashion subsequent to activation intopro-inflammatory DCs. Thus, the method represents a large scale andcost-effective clinical grade production method of non-exhaustedimmature dendritic cells.

A further aspect of the invention relates to a mixture of allogeneicnon-exhausted immature dendritic cells (DCs) originating from at leasttwo different, allogeneic donors. Such a mixture is obtainable by thedescribed method.

A further aspect of the invention relates a method of producingpro-inflammatory DCs. By activating the non-exhausted immature DCspro-inflammatory DCs are obtained. By such a method, a mixture ofallogeneic pro-inflammatory dendritic cells originating from at leasttwo different, allogeneic donors is obtainable. The mixture may beformulated into pharmaceutical composition further comprising at leastone pharmaceutical acceptable carrier. The mixture and thepharmaceutical composition, respectively, may be used the treatment ofcancer.

Further advantageous features of the invention are defined in thedependent claims. In addition, advantageous features of the inventionare elaborated in embodiments disclosed herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present inventors did envisage that monocyte-containing leukocytepopulations which are present in buffy coats or trapped in leukocyteremoval filters used for depleting leukocytes from whole blood (used forenrichment of red blood cells) or leukocyte removal filters used fordepleting leukocytes from pooled buffy coats (used for enrichments ofplatelets), could potentially be used for large-scale and cost-effectiveproduction of non-exhausted immature dendritic cells.

As previously shown, leukocytes retained by different types of leukocyteremoval filters can be recovered by back-flushing with a suitable mediumfollowed by monocyte enrichment (Ebner S et al., Generation of largenumbers of human dendritic cells from whole blood passaged throughleukocyte removal filters: an alternative to standard buffy coats JImmunol Methods 252 (2001) 93-104; and Meyer T P H et al., Filter BuffyCoats (FBC): A source of peripheral blood leukocytes recovered fromleukocyte depletion filters. J Immunol Methods 307 (2005) 150-166).

A buffy coat is the fraction of an anti-coagulated blood sample thatcontains most of the leukocytes, including neutrophils, basophils,eosinophils, monocytes, and lymphocytes, and platelets following densitygradient centrifugation of whole blood. Buffy coats are normally used asraw material for platelet production. During this process, leukocytereduction by a leukocyte depletion filter is performed after pooling of4 to 8 buffy coats. Typically, the TACSI equipment or theOrbiSac systemis used for platelet isolation from buffy coats.

The TACSI equipment for platelet preparation from pooled buffy coatscontains a system box (fixed on the rotor) and an insert that can beremoved for the mounting of the TACSI kit. Each system box is providedwith a press system, controlled and monitored by an individualmicroprocessor. In a first sequence, pooled buffy coats within thepooling container are sedimented by centrifugation in a verticalposition. In the following step, the platelet-rich layer (alsocontaining a substantial amount of leukocytes, including monocytes andlymphocytes) of the pooled buffy coat supernatant is transferred into astorage container by the activation of the press system in each box. Inaddition, the filter for leukodepletion is integrated in the TACSI kitbetween the processing bag and the final storage container. Theleukocyte depletion filter and the rest of the buffy coat within thebuffy coat pooling container, both containing substantial amount ofmonocytes, are then discharged.

In the alternative OrbiSac system for automated platelet enrichment, thebuffy coat pooling container is ring-shaped. After centrifugation, theplatelet-rich central part of the supernatant is transferred into acontainer placed in the center of the centrifuge and the transfer ismade through an integrated leukocyte depeletion filter. The leukocytedepletion filter and the rest of the buffy coat within the buffy coatpooling container, both containing substantial amount of monocytes, isthen discharged.

In summary, methods in the art for platelet enrichment from buffy coatsprovide two possible sources of monocytes, i.e. the rest of the buffycoat being depleted of platelets, also denoted platelet depleted buffycoat, and the leukocyte depletion filter. The platelet depleted buffycoat and the leukocyte depletion filter each contains a mixture ofleukocytes, including up to 1 billion monocytes. However, thesemonocytes are allogeneic with respect to each other, as they origin fromdifferent, allogeneic donors, due to the pooling of buffy coat prior tothe platelet depletion.

Un-pooled buffy coats, containing platelets, or filters obtained afterleukocyte depletion of whole blood will maximally only provide up toabout 100 to 200 millions monocytes per buffy coat or filter. Thereforethey must be pooled in order to provide a number sufficient forcost-effective GMP-production of non-exhausted immature DCs.

Similar to leukocytes obtained from platelet production pooledleukocytes from buffy coats, containing platelets, or from filters usedto deplete whole blood from leukocyte will also consist of a mixed cellpopulation originating from different allogeneic donors.

Pooling of leukocytes from at least 5 to 10 buffy coats or pooling ofeluted leukocytes from at least 5 to 10 whole blood leukocyte filterswould, at least theoretically, solve the problem of providing asufficient number of leukocytes for large-scale and cost-effectiveclinical grade production of non-exhausted immature DCs.

Similarly, pooled, platelet depleted buffy coats and/or leukocytedepletion filters used for platelet enrichment from buffy coats, canpotentially be used to provide a sufficient number of leukocytes forlarge-scale and cost-effective clinical grade production ofnon-exhausted immature DCs.

As previously shown, leukocytes retained by leukocyte depletion filterscan be recovered by back-flushing with a suitable medium followed bymonocyte enrichment (Ebner S et al., Generation of large numbers ofhuman dendritic cells from whole blood passaged through leukocyteremoval filters: an alternative to standard buffy coats J ImmunolMethods 252 (2001) 93-104; and Meyer T P H et al., Filter Buffy Coats(FBC): A source of peripheral blood leukocytes recovered from leukocytedepletion filters. J Immunol Methods 307 (2005) 150-166).

However, an envisaged problem associated with the subsequentco-culturing of monocyte-enriched leukocytes, which are derived fromdifferent, allogeneic donors, is that their incompatibility as to majorhistocompatibility complex (MHC) class I and class II antigens is deemedto lead to a premature activation of monocytes/immature DCs from onedonor by contaminating alloreactive T cells and/or natural killer cellsfrom another donor.

Co-culturing in standard cell culture medium, such as RPMI-1640(RPMI=Roswell Park Memorial Institute, at which institute the mediumoriginal was developed by Moore et. al.) with fetal calf serum, or inserum-free cell culture medium, such as X-VIVO 15, of mononuclear cells,including monocytes, lymphocytes and NK-cells, from two allogeneicdonors is known to induce production of well-known DC-activatingfactors, including TNF-alpha (Laurin et al, Transplantation 2004;77:267; Wallgren et al, Scand J Immunol 2005; 62:234). Further, additionof culture supernatants from such co-cultures in standard RPMI-1640medium with fetal calf serum or standard serum-free medium of allogeneicmononuclear cells have repeatedly been shown to induceactivation/maturation of monocyte-derived, immature, DCs (Laurin et al,Transplantation 2004; 77:267; Wallgren et al, Scand J Immunol 2005;62:234).

Furthermore, addition of TNF-alpha to standard RPMI-1640 mediasupplemented with GM-CSF and IL-4 used for differentiation of monocytesinto immature DCs has been shown to induce premature activation andsubsequent exhaustion (tolerance) of differentiated DCs (Rieser C etal., Differential Deactivation of Human Dendritic Cells by EndotoxinDesensitization: Role of Tumor Necrosis Factor-α and Prostaglandin E2.Blood 91 (1998) 3112-3117).

The problem with contaminating T cells and NK cells is of relevance evenafter GMP-enrichment (elutriation and antibody/bead isolation) ofmonocytes from pooled buffy coats or from leukocyte deletion filters(Schwanke et al, Journal of Clinical Apheresis 21: 153-157 (2006); Meyeret al, Journal of Immunological Methods 307 (2005) 150-166) due to thedifficulty in preparing monocyte cell populations essentially free fromcontaminating T cells and NK cells.

Furthermore and importantly, not only co-culturing with allogeneiclymphocytes in standard medium, but also co-culturing of monocytes withallogeneic neutrophils in RPMI-1640 media supplemented with fetal calfserum, results in up-regulation of membrane CD40, CD86, and humanleukocyte antigen (HLA)-DR on DCs, i.e. premature activation, as hasbeen shown by Meggiovanni et al (cf. Journal of Leukocyte Biology, 2006;79; 977-988). Substantial removal of neutrophils from monocytes withinpooled buffy coats or eluted filter leukocyes using elutriation(Schwanke et al, Journal of Clinical Apheresis 21: 153-157 (2006)) oranti-body/bead isolation (Meyer et al, Journal of Immunological Methods307 (2005) 150-166) has been shown to be very difficult. Usually such anmonocyte-enriched product contains a significant amount (i.e. 25-40%based on the total number of cells present) of neutrophils. From asafety perspective this neutrophil contamination is however not aproblem.

Moreover, even if it was possible to prepare 100% pure monocyte cellpopulations, this would not eliminate the risk of premature activationdue to active interactions between monocytes from different, allogeneicdonors. In a recent review paper entitled “Origin and biology of theallogeneic response”, by the distinguished and renowned immunologistsFadi G. Lakkis and Robert I. Lechler (cf. Cold Spring Harborperspectives in medicine, Vol. 3, No. 8, 2013) it was conclude that aninnate allorecognition mechanisms indeed exist. The authors state that:“Allograft rejection is not restricted to vertebrate animals endowedwith adaptive immune systems, but is common to many invertebrateorganisms that predate the evolution of adaptive immunity (animals thatlack T and B lymphocytes, NK cells, somatic gene rearrangement enzymes,and the MHC)”.

Furthermore, and perhaps more direct, allogenic responses are seen alsoin mice devoid of lymphoid cells. Activation of monocytes is dependenton differences in non-MHC antigens between recipient monocytes andinjected allogeneic donor leukocytes, including allogeneic monocytes(Zecher D et al., An Innate Response to Allogeneic Nonself Mediated byMonocytes. J Immunol 83 (2009) 7810-7816). Zecher et al. showed thatinjecting allogeneic leukocytes into the ear pinnae of RAG2/2 mice,lacking T and B lymphocytes, elicits significantly greater swelling andinfiltration of the skin with host myeloid cells than injectingsyngeneic leukocytes. The response to the allogeneic leukocytes occurredindependently of NK cells and was mediated by monocytes. Further, themonocyte response was to allodeterminants not linked to the MHC.

In an even more recent paper (Zeng Q, et al. “Innate recognition ofallogeneic non-self induces monocyte differentiation to mature dendriticcells in vivo.” Am J Transplant 12: 148-148, 2012), the authors showedthat heart allografts transplanted to gc2/2RAG2/2 mice, which lack T, B,and NK cells, are rapidly infiltrated by host monocytes thatdifferentiate into mature, IL-12-expressing dendritic cells (DCs). Thedeterminants on allogeneic cells that trigger host monocyte maturation,and the putative monocyte receptors that recognize them, are however notknown yet.

There is thus clear evidence that mammalian monocytes directly respondto non-MHC determinants on allogeneic cells independently of T, B, andNK cells. Hence, according to the generally prevailing perception,alloreactivity is deemed to be a general property of monocytes.

Taken together, this implies that co-culturing of monocyte-enriched cellpopulations derived from different allogeneic donors clearly isenvisaged to lead to pre-activation and subsequent exhaustion of themonocytes during their differentiation into monocyte-derived DCs.Thereby the DCs will be unable to become PI-DCs producing requiredadjuvant factors in a sustained fashion when re-stimulated with relevantactivating factors.

This envisaged activation-induced exhaustion is similar to thewell-known exhaustion of monocytes, macrophages and DCs that is inducedby a premature activation with inflammatory agents like TNF-α (Park etal, Nat Immunol. 2012; 12: 607-615) or microbial lipopolysaccharides(LPS) (Rieser C et al., Differential Deactivation of Human DendriticCells by Endotoxin Desensitization: Role of Tumor Necrosis Factor-α andProstaglandin E2. Blood 91 (1998) 3112-3117; Langenkamp A et al.,Kinetics of dendritic cell activation: impact on priming TH1, TH2 andnonpolarized T cells. Nature Immunol. 1 (2000) 311-316;).

The present inventors have however surprisingly found that non-exhaustedimmature DCs actually can be propagated from an initial cell populationconsisting of a mixture of allogeneic monocyte-enriched leukocytes fromdifferent, allogeneic donors, in a manner similar to the one used topropagate non-exhausted immature DCs from enriched monocytes which arederived from one single donor (cf. WO 2011/098516), i.e. by use of anaqueous cell culture medium free from non-human serum, but supplementedwith interleukin-4 (IL-4) and granulocyte-macrophage colony stimulatingfactor (GM-CSF).

In contrast to the prevailing prejudice, propagation of a mixture ofenriched monocytes from different, allogeneic donors into immature DCsunder certain conditions was surprisingly shown to not result inpremature activation and subsequent exhaustion of the DCs. Theseconditions include co-culturing in aqueous cell culture medium free fromnon-human serum and supplemented with GM-CSF and IL-4.

However, co-culturing in aqueous cell culture medium free from non-humanserum and not supplemented with interleukin-4 (IL-4) andgranulocyte-macrophage colony stimulating, as well as co-culturing inaqueous cell culture medium comprising non-human serum, e.g. bovine calfserum, and supplemented with interleukin-4 (IL-4) andgranulocyte-macrophage colony do, as expected and recognized in the art,result in premature activation and subsequent exhaustion of the DCs.

Thus, monocytes may be isolated from pooled leukocyte populations fromdifferent allogeneic donors, making it possible to perform large-scaleand cost-effective GMP-enrichment of monocytes, such as elutriation(Elutra) or antibody/bead-isolation (CliniMacs). Subsequently,non-exhausted immature DCs can be generated from the isolatedmonocyte-enriched allogeneic leukocytes without premature activation.

Such non-exhausted immature DCs may be used to propagatepro-inflammatory-DCs which are aimed to function as an anti-tumorvaccine when injected intratumorally (cf. WO 2011/098516). Further,non-exhausted immature DCs from different, allogeneic donors, may beloaded with tumor antigen(s) before activation in order to produce a“complete” cellular allogeneic anti-cancer vaccine (cf. EP 1 509 244 B1)that can be injected into different sites, including intratumoral,subcutaneous, epicutaneous, intramuscular and/or intravenous sites.

An embodiment thus relates to a method for producing non-exhaustedimmature DCs from a mixture of monocyte-enriched allogeneic leukocytes.In such a method, a mixture of allogenic leukocytes, obtained from leasttwo different, allogeneic donors, is provided. According to anembodiment, two different, allogeneic donors are intended to mean thatthe two individuals donating leukocytes, are of the same species but ofdifferent genetic constitution, i.e. antigenically distinct. As alreadydescribed, allogenic leukocytes may be obtained from pooled buffy coatsor by eluting leukocytes from used leukocyte-depletion filters.Allogeneic monocytes are then isolated from the provided mixture ofallogeneic leukocytes. Subsequently, non-exhausted immature DCs aregenerated from the isolated monocyte-enriched allogeneic leukocytes.

Except for the ability to perform large-scale and cost-effectiveGMP-enrichment of monocytes, a further advantage of non-exhaustedimmature DCs, originating from a mixture of allogeneic monocytes derivedfrom at least two different allogeneic donors, is that the normalbiological variation as to production of different pro-inflammatoryfactors upon activation, known to exist between PI-DCs from differentdonors, will be reduced.

In a preferred embodiment, the allogenic leukocytes are provided bypooling of at least two buffy coats, comprising leukocytes. The buffycoats to be pooled are obtained from at least two different, allogeneicdonors. The pooled buffy coats may contain platelets or they may beplatelet depleted.

Allogeneic leukocytes may also be provided by eluting leukocytes from atleast two leukocyte depletion filters, which filters, respectively,previously have been used to deplete leukocytes from whole blood, fromat least two different allogeneic donors. After the elution, theobtained leukocytes are pooled to obtain a mixture of allogeneicleukocytes. Evidently, but less preferred, the whole blood may also bepooled prior to leukocyte depletion. A procedure for eluting leukocytesfrom a depletion filter, which filter previously has been used toeliminate leukocytes from whole blood, has been described by Ebner et al(cf. Journal of Immunological Methods 252 (2001) 93-104).

Similarly, allogeneic leukocytes may also be provided by elutingleukocytes from a leukocyte depletion filter, which filter has been usedto deplete leukocytes from pooled buffy coats, wherein the pooled buffycoats originate from at least two different allogeneic donors. Aprocedure for eluting leukocytes from a depletion filter, which filterpreviously has been to eliminate leukocytes from a buffy coat have beendescribed by Meyer et al (cf. Journal of Immunological Methods 307(2005) 150-166).

While such allogeneic leukocytes, obtained from leukocyte depletionfilter, also may be used to produce non-exhausted immature DCs, it seemsthat is preferred to use allogenic leukocytes provided by pooling of atleast two buffy coats, obtained from at least two different, allogeneicdonors. Allogenic leukocytes eluted from leukocyte depletion filter, mayproduce somewhat lower amounts of chemokines, except for MIG, andcytokines, subsequent to maturation, as compared to allogeneic monocytesderived directly from pooled peripheral blood samples or from pooledbuffy coats

Isolation of monocytes from a mixture of different leukocytes iswell-known in the art. According to an embodiment, monocytes areisolated from the provided mixture of allogeneic leukocytes byestablished GMP-production methods. Thus, monocytes may be isolated fromthe provided mixture of allogeneic leukocytes by elutriation or byantibody/bead isolation.

Elutriation is a technique wherein continuous counter-flow elutriationseparates cells into multiple fractions. In short, a constantcentrifugal force that separates the cells by density counters acontinuously increasing media flow streaming through the sedimentdispersing the cells by size. Hence, smallest/lightest first andbiggest/heaviest last, the media flow flushes the cells away intoseveral products.

Antibody/bead isolation of monocytes is performed by (Immuno)-magneticactivated cell sorting (MACS). MACS is a widely employed technique forselective isolation of cells from whole blood, buffy coats, or WBCapheresates. In short, CD-specific antibodies bearing ferro-magneticbeads at their Fc-terminus are coupled to wanted (positive selection) orunwanted (negative selection) cells. Such treated cells may be retainedwithin a porous, metal coated column when exposed to a strong magneticfield.

Subsequent to the isolation, the monocytes are differentiated intoimmature DCs, i.e. immature DCs are generated. Immature DCs aregenerated by co-culturing the allogeneic monocytes in an aqueous cellculture medium free from non-human serum and supplemented withgranulocyte-macrophage colony stimulating factor (GM-CSF) in combinationwith interleukin-4 (IL-4), for 2 to 7 days, such as about 5 days,thereby differentiating the monocytes into immature DCs.

As cell culture media comprising fetal calf serum was found to inducepremature activation despite being supplemented with GM-CSF and IL-4, itis important that the medium used is free from non-human serum.

Immature DCs may also be generated by culturing the allogeneic monocytesin an aqueous media comprising GM-CSF in combination with interleukin-2(IL-2), interleukin-15 (IL-15) or interferon alpha for 2 to 7 days, suchas 5 days, thereby differentiating the monocytes into immature DCs. Useof GM-CSF in combination with IL-4 is however preferred as it has beenshown to prevent alloreactivity and premature activation, when used incombination with a medium free from non-human serum.

The person skilled in the art is familiar with cell culture media andtheir components. Typically, the cell culture medium used comprises:

at least one salt, such as NaCl, KCl, MgSO₄, and/or Ca(NO₃)₂;

at least one sugar, such as glucose;

one or several amino acid(s), such as L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophane, L-tyrosine, L-valine,L-arginine, L-asparagine, L-aspartic, L-cystine, L-glutamine, L-glutamicacid, glycine, L-histidine, L-hydroxyproline, L-isoleucine, L-leucine,and/or L-lysine;

one or several vitamin(s) and other vital nutrient(s), such asglutathione, biotin, vitamin B12, D-Ca-pantothenate, cholin chloride,folic acid, myo-inositol, nictoninamid, p-amino benzoic acid, pyridoxin,riboflavin, and/or thiamine; and

at least one buffer, such as phosphate salt (e.g. Na₂HPO₄) and/or acarbonate salt (e.g. NaHCO₃).

According to an embodiment, the culture medium comprises at least onesalt, such as NaCl, at least one sugar, such as glucose, one or severalamino acid(s), one or several vitamin(s), and a buffer, such asphosphate salt (e.g. Na₂HPO₄) and/or a carbonate salt (e.g. NaHCO₃).

Further, while the cell culture medium further is free from non-humanserum it typically comprises at least human polypeptide. According to anembodiment, the cell culture medium comprises at least one humanpolypeptide selected from the group consisting of transferrin, albumin,and insulin; preferably the cell culture medium comprises all three ofthem. The human polypeptide may be obtained from human plasma. Furtherthey may be recombinantly produced. As an example insulin may berecombinantly produced in yeast cells.

As an example, the cell culture medium free from non-human serum may beCellGro®, which is a GMP serum-free dendritic dell medium (DC) providedby CellGenix GmbH. In the US the medium is sold under the trademarkCellGenix™

As recognized by the skilled person and as explained herein, the termisolated does not necessarily refer to 100% purity, but to monocytesobtained by an isolation process showing preference for monocytes.Monocytes obtained by such a process may be referred to asmonocyte-enriched allogeneic leukocytes, as other leukocytes in additionto monocytes will be present.

According to an embodiment, the allogeneic monocytes are enriched fromthe mixture of allogeneic leukocytes. Monocyte-enriched allogeneicleukocytes in addition to monocytes typically also comprise allogeneicneutrophils. Further, they may comprise other granulocytes.

In contrast to the prevailing prejudice, no signs of prematureactivation was seen, when the allogenic monocytes were co-cultured inaqueous cell culture medium free from non-human serum and supplementedwith GM-CSF/IL-4, despite that fact that immature DCs were obtained froma mixture of allogeneic leukocytes. Accordingly, such immature DCs arenon-exhausted, thus being able to produce substantial amounts, such asmore than 2 000, 5 000, or 7 500 pg/mL, of pro-inflammatory chemokines,including MIP-1 alpha, MIP-1beta, RANTES and MIG, and substantialamounts, such as more than 500, 1 500 or 3 000 pg/mL, ofpro-inflammatory cytokines, including IL-12p70 and TNF-alpha in asustained fashion subsequent to withdrawal of the activating factors.

According to an embodiment, immature is intended to mean DCs whichexpress only low levels of the DC maturation markers CD83 and CD86 andwhich are able to produce high amounts of proinflammatry chemokines andcytokines upon activation. Low levels are, according to embodiment, tobe interpreted such that an at least 3-fold, such as at least 5-fold,increase in the CD83-expression is seen upon activation, and that an atleast 5-fold, such as at least 8-fold, increase in the CD86-expressionis seen upon activation.

As it was envisaged that premature activation was to be seen, thecycloxoygenase-2 inhibitor NS-398, a factor known to hamperprostaglandin E2 (PGE2)-mediated exhaustion of activated DCs was addedin some experiments. However, the presence of NS-398 during propagationof monocytes into DCs did not increase, but rather decrease, theactivation-induced production of MIG and IL-12p70. Thus, there are nosigns of PGE2-mediated exhaustion of differentiated immature DCs fromco-cultures of mixed allogeneic monocytes.

As recognized by the skilled person (cf. e.g. EP 1 509 244 B1 and WO2011/098516), non-exhausted immature dendritic cells (DCs) are useful inthe production of pharmaceutical composition for the treatment ofcancer. Thus, an embodiment relates to a mixture of allogeneicnon-exhausted immature dendritic cells (DCs) originating from at leasttwo different, allogeneic donors. Such dendritic cells (DCs) areobtainable by such a method as disclosed herein. Subsequent to thedifferentiation into immature DCs, the immature DCs may be activated tobecome pro-inflammatory DCs. Activation may be induced in several ways.Many signals have been shown to induce at least some aspects of DCactivation. Among the most powerful of these are microbial and viralproducts (pathogen-associated molecular patterns (PAMPs), which aredirectly recognized by pattern-recognition receptors (PRRs), includingmembers of the Toll-like receptor (TLR) family. PRRs control theexpression of many innate response genes and can directly signal for DCactivation. In addition, PRR signaling in both immune and non-immunecells often leads to the synthesis of inflammatory cytokines, such astumor necrosis factor (TNF) and interleukin 1 (IL-1), which can alsopromote DC activation. Thus, addition of inflammatory cytokines may alsocontribute to the activation of immature DCs.

According to an embodiment, the immature DCs are loaded with antigensprior to, or simultaneous with, the activation, in order to provide acellular allogeneic anti-cancer vaccine. Antigen-loading is well-knownin the art (cf. e.g. EP 1 509 244 B1) and may performed with methods,such as pulsing, transfection, infection or fusion. As an example, theantigen may typically be obtained from a tumor; typically the tumor typewhich the vaccine is to be directed to. In obtaining antigens, arepresentative specimen of cancer type of interest typically is used.

According to a preferred embodiment, the activation of the immature DCsis performed in accordance with the method disclosed in WO 2011/098516.Maturation may thus be induced by adding the Toll-like receptor 3(TLR3)-ligand poly-I:C, a TLR7/8-ligand, such as R848 (Resiquimod) andthe cytokine interferon gamma (IFN-γ). The Toll-like receptor 3(TLR3)-ligand poly-I:C is a synthetic analog of dsRNA comprising astrand of poly(I) annealed to a strand of poly(C). The size of thestrand may vary. The size may be 200 base pairs to 8 000 base pairs,such 200 to 1 500 or 1 500 to 8 000 base pairs. The TLR7/8-ligand R848is also denoted Resiquimod in the art. As an alternative to Resiquimod,Gardiquimod or Imiquimod may be used as TLR7/8-ligands. Typically, theimmature DCs are exposed to the activation factors for 8 to 24 hours,such as 18 hours.

The activation may further include the addition of at least onesubstance selected from the group consisting of TLR2-ligands,TLR4-ligands, such as bacterial lipopolysaccharide and monophosphoryllipid A, TLR9-ligands, such as CpG oligonucleotides (ODN) sequences thatdistinguish microbial DNA from mammalian DNA, Interferon alpha (IFN-α),interleukin 1β (IL-1β), and tumor necrosis factor alpha (TNF-α).Further, the activation does preferably not comprise addition ofprostaglandin E2 (PGE2) in order to prevent the mature DCs from becomingmigratory DCs that rapidly will leave the injection site (tumor), whichwould be disadvantageous within the context of this invention.

Subsequent to the activation, the resulting pro-inflammatory DCs may bewashed to remove essentially all of the activation factors. Thus, theactivation factors typically are washed away prior to use of thepro-inflammatory DCs as vaccine. Removal of the activation factorsavoids co-administration of activation factors (aimed to inducepro-inflammatory DCs ex vivo). Co-administration of activation factorsmost likely will lead to a strong and persistent activation also ofintratumorally recruited immature DCs, leading to their differentiationinto pro-inflammatory mature DCs rather than the desired differentiationinto migratory mature DCs.

As already described (cf. WO 2011/098516), pro-inflammatory dendriticcells are useful in the treatment of cancer, as they may activate apatients own DCs to develop into tumor-loaded migratory DCs. Anembodiment, thus relates to mixture of allogeneic pro-inflammatorydendritic cells originating from at least two different, allogeneicdonors. Such allogeneic pro-inflammatory dendritic cells are obtainableby such a method as disclosed herein. By freezing the pro-inflammatorydendritic cells subsequent to the activation they may be stored.Typically the pro-inflammatory dendritic cells are frozen in a mediumcontaining dimethylsulphoxide (DMSO) and serum or plasma. Before use,the frozen cells are thawed and the DMSO is washed away.

For use in the treatment of cancer, such allogeneic pro-inflammatorydendritic cells may be formulated into a pharmaceutical composition. Thepharmaceutical composition may comprise at least one pharmaceuticalacceptable carrier, such as a phosphate buffered saline solution, water,and emulsions, such as an oil/water or water/oil emulsion, and varioustypes of wetting agents. Further, it may comprise pharmaceuticalacceptable adjuvants, excipients, stabilizers preservatives and/or othercomponents known in the art. As an example, the carrier may be a salinesolution comprising human serum albumin.

A further embodiment relates to such a mixture of allogeneicpro-inflammatory dendritic cells, or such a composition comprising suchallogeneic pro-inflammatory dendritic cells, for use in the treatment ofcancer. Similarly, an embodiment relates to use of such a mixture ofallogeneic pro-inflammatory dendritic cells for use in the manufactureof a medicament for the treatment of cancer. A further embodimentrelates to a method of treating cancer, wherein a mixture of allogeneicpro-inflammatory dendritic cells is administrated to a patient in needof such treatment in dose sufficient to activate the patients own DCs todevelop into tumor-loaded migratory DCs.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description and the following experimentalpart, utilize the present invention to its fullest extent. The preferredspecific embodiments described herein are, therefore, to be construed asmerely illustrative and not limitative of the remainder of thedescription in any way whatsoever. Further, although the presentinvention has been described above with reference to specificembodiments, it is not intended to be limited to the specific form setforth herein. Rather, the invention is limited only by the accompanyingclaims and, other embodiments than the specific above are equallypossible within the scope of these appended claims, e.g. different thanthose described above.

In the claims, the term “comprises/comprising” does not exclude thepresence of other elements or steps. Additionally, although individualfeatures may be included in different claims, these may possiblyadvantageously be combined, and the inclusion in different claims doesnot imply that a combination of features is not feasible and/oradvantageous.

In addition, singular references do not exclude a plurality. The terms“a”, “an”, “first”, “second” etc do not preclude a plurality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrate the expression of the activation/maturation markersCD86 and CD83 on immature DCs and PI-DCs derived from single or mixedperipheral blood monocyte cultures.

FIG. 2 illustrates pro-inflammatory chemokine production by immature DCs(derived from single or mixed peripheral blood monocyte cultures) during18 hours of persistent stimulation with activating factors (“Activeproduction”).

FIG. 3 illustrates pro-inflammatory cytokine production by immature DCs(derived from single or mixed peripheral blood monocyte cultures) during18 hours of persistent stimulation with activating factors (“Activeproduktion”)

FIG. 4 illustrates pro-inflammatory cytokine production by immature DCs(derived from single or mixed peripheral blood monocyte cultures) during18 hours of persistent stimulation with activating factors+/−addition ofthe cycloogygenase-2 (Cox-2) inhibitor NS-398

FIG. 5 illustrates pro-inflammatory chemokine production by immature DCs(derived from single or mixed buffy coat monocyte cultures) during 18hours of persistent stimulation with activating factors (“Activeproduction”)

FIG. 6 illustrates pro-inflammatory cytokine production by immature DCs(derived from single or mixed buffy coat monocyte cultures) during 18hours of persistent stimulation with activating factors (“Activeproduction”)

FIG. 7 illustrates pro-inflammatory chemokine production by PI-DCs(derived from single or mixed peripheral blood monocyte cultures). ThesePI-DCs have been washed after stimulation with activating factors for 18hours and subsequently re-cultured for 24 hours without addition ofactivating factors (“Passive production”).

FIG. 8 illustrates pro-inflammatory cytokine production by PI-DCs(derived from single or mixed peripheral blood monocyte cultures). ThesePI-DCs have been washed after stimulation with activating factors for 18hours and subsequently re-cultured for 24 hours without addition ofactivating factors (“Passive production”).

FIG. 9 illustrates pro-inflammatory chemokine production by PI-DCs(derived from single or mixed buffy coat monocyte cultures). ThesePI-DCs have been washed after stimulation with activating factors for 18hours and subsequently re-cultured for 24 hours without addition ofactivating factors (“Passive production”).

FIG. 10 illustrates pro-inflammatory cytokine production by PI-DCs(derived from single or mixed buffy coat monocyte cultures). ThesePI-DCs have been washed after stimulation with activating factors for 18hours and subsequently re-cultured for 24 hours without addition ofactivating factors (“Passive production”).

FIG. 11 illustrates that mixed immature DCs derived from filtermonocytes produce substantial amounts of pro-inflammatory chemokinesduring 18 hours of persistent stimulation with activating factors(“Active production”)

FIG. 12 illustrates that mixed immature DCs derived from filtermonocytes produce substantial amounts of pro-inflammatory cytokinesduring 18 hours of persistent stimulation with activating factors(“Active production”)

FIG. 13 illustrates that mixed PI-DCs derived from filter monocytesexhibit a substantial production of pro-inflammatory chemokines afterwithdrawal of activating factors. These PI-DCs have been washed afterstimulation with activating factors for 18 hours and subsequentlyre-cultured for 24 hours without addition of activating factors(“Passive production”).

FIG. 14 illustrates that mixed PI-DCs derived from filter monocytesexhibit a substantial production of pro-inflammatory cytokines afterwithdrawal of activating factors. These PI-DCs have been washed afterstimulation with activating factors for 18 hours and subsequentlyre-cultured for 24 hours without addition of activating factors(“Passive production”).

EXPERIMENTAL

The following examples are mere examples and should by no mean beinterpreted to limit the scope of the invention. Rather, the inventionis limited only by the accompanying claims.

Leukocyte of Various Origin

Isolation of Leukocytes from Leukocyte Depletion Filters (TACSI Filters)

Leukocyte filters (TACSI leukocyte depletion filters used for routineleukocyte depletion of 4 pooled buffy coats during platelet production)were collected at the Component Laboratory at the Department ofTransfusion Medicine, Sahlgrenska University Hospital, Gothenburg, andtransported to the laboratory (Department of Clinical Immunology,Sahlgrenska University Hospital) on ice.

In the laboratory, a Syringe (Terumo) was filled with 50 ml of PBS/EDTAbuffer (CliniMACS) and connected to the TACSI filter through a luer-lockfitting. The filter was back-flushed into a sterile glass flask, threetimes (150 ml PBS/EDTA buffer in total). The eluted cell suspension wasfinally diluted with PBS (PAA, Fisher Scientific) at a 1:2 concentrationin a Falcon tube (Fisher brand, Fisher Scientific).

Buffy Coats

Buffy coats from healthy blood donors were collected at the departmentof Transfusion Medicine and transported to the laboratory at roomtemperature.

Peripheral Blood

Peripheral blood from healthy donors was collected at the department ofTransfusion Medicine and transported to the laboratory at roomtemperature. In the laboratory, the blood was mixed with roomtemperature PBS at a 1:2 concentration in a Falcon tube.

Isolation of Peripheral Blood Mononuclear Cells (PBMC)

Peripheral blood from healthy donors was collected at the department ofTransfusion Medicine and transported to the laboratory at roomtemperature. In the laboratory, the blood was mixed with roomtemperature PBS at a 1:2 concentration in a Falcon tube. The cellsuspension was gently transferred to 10 ml centrifuge tubes (Nunc)containing 3 ml of Lymphoprep (Axis-Shield). 5-6 ml was transferred toeach tube followed by centrifugation at 2000 rpm, 20 min at roomtemperature and without brake. The isolated PBMCs were transferred topre-cooled 10 ml tubes. The cells were washed twice by filling the tubeswith cold PBS followed by centrifugation at 1450 rpm, 10 minutes at 4°C. The supernatants were discarded and the pellets were re-suspended in1 ml of cold PBS. Another 9 ml of was added to each tube.

Monocyte Isolation

5 mL fractions of eluted filter leukocytes/buffy coat leukocytes orisolated PBMCs from 10-20 mL of whole peripheral blood were centrifugedin tubes at 1450 rpm, 10 minutes at 4° C. The supernatants werecompletely removed and the cell pellets were re-suspended in 80 μl ofPBS/EDTA (Miltenyi) per 10⁷ cells. 20 μl of CD14 microbeads (Miltenyi)was added per 10⁷ cells. The cells were mixed and incubated for 15minutes at 4° C. and subsequently washed by adding 1-2 ml of PBS/EDTAfollowed by centrifugation at 300×g for 10 minutes. The supernatantswere completely removed and remaining cells were re-suspended in 500 μlof PBS/EDTA.

MidiMACS separators (Miltenyi) were placed in a magnetic multistand(Miltenyi) and rinsed with 3 ml of PBS/EDTA. The cell suspensions wereplaced onto the MidiMACS separators allowing the cells to pass through.The MidiMACS separators were washed three times with 3 ml of PBS/EDTA.The effluent fractions with unlabeled cells were discarded. The MidiMACSseparators were removed from the magnetic multistand and placed onto aFalcon tube. 5 ml of PBS/EDTA buffer was pipetted onto the column andthe cells were immediately pushed through with a plunger.

Cell concentration was determined in a Bürker chamber. The cellsuspensions containing monocytes were centrifuged at 1450 rpm, 10minutes at 4° C. The supernatants were discarded and the cells werere-suspended in CellGro DC-media (CellGenix). The purity of CD14+monocytes within all monocyte-isolated cell cultures was >80%, asdetermined by FACS-analysis, see below.

Generation of Immature DCs

The leukocytes originating from the TACSI filters were re-suspended tothe concentration of 300 000 cells/mL in CellGro DC-media, being amedium free from non-human serum, and plated in 24-well plates (1 mL perwell). Monocyte-enriched leukocytes from buffy coats and peripheralblood were first re-suspended to a concentration of 5×10⁵ monocytes/mLin CellGro media. 400 μl of CellGro media (whithout cells) was firstadded to 12 wells (A1-6, B1-3, C1-3) in a 24-well plate. 600 μl of themonocyte-enriched cell suspension from donor A (buffy coat or peripheralblood respectively) was transferred to well A1-3. 600 μl ofmonocyte-enriched cell suspension from donor B (buffy coat or peripheralblood) was transferred to well B1-3. 600 μl of the monocyte-enrichedcell suspension from donor C was transferred to well C1-3 (buffy coat orperipheral blood). In well A4-6, 200 μl of monocyte-enriched cellsuspension was transferred from all three donors (buffy coat orperipheral blood). The final cell number in all wells was 300 000 cells(in a volume of 1 mL CellGro media per well).

In order to differentiate the monocytes into immature DCs, the culturemedium was supplemented with 1000 U/mL recombinant human IL-4 and 1000U/mL recombinant human GM-CSF (all from CellGenix, Freiburg, Germany)and cells were subsequently cultured for 5 days.

Activation/Maturation of Immature DCs

Following 5 days of culture in CellGro medium supplemented with IL-4 andGM-CSF, activation/maturation of the immature DCs was induced by adding20 μg/mL polyI:C (Sigma, Steinheim, Germany), an immunostimulantspecific to the TLR-3 receptor also known as polyinosinic:polycytidylicacid or polyinosinic-polycytidylicf acid sodium salt, 2.5μg/mL R848(Sigma, Steinheim, Germany), toll-like receptor 7/8-ligand also known asresiquimod, and 1000 U/ml interferon gamma (IFN-γ, R&D systems,Minneapolis, USA). After 18 h of incubation, the cells were washed threetimes and further incubated in fresh AIM-V medium (without addition ofexogenous activating factors) for 24 h Culture supernatants from thecultures were harvested according to protocols well known to a personskilled in the art.

ELISA analysis was performed on the supernatants as described below, inorder to analysis the levels of pro-inflammatory chemokines and thepro-inflammatory cytokines

Evaluation of the Levels of Pro-Inflammatory Chemokines and thePro-Inflammatory Cytokines by ELISA

The pro-inflammatory chemokines CCL3/MIP-la, CCL4/MIP-1β, CCL5/RANTESand CXCL9/MIG and the pro-inflammatory cytokines IL-12p70 and TNF-α weremeasured by enzyme-linked immune adsorbent assay (ELISA) using Duo SetELISA Development System from R&D systems, Minneapolis, USA according tothe manufacturers instructions.

Phenotypic Examination by Flow Cytometry

Monocytes and monocyte-derived DCs were generated as described above.The frequency of CD14+ monocytes after monocyte isolation was estimatedby staining cells with FITC-anti human CD14. After 5 days of incubationin CellGro supplemented with IL-4 and GM-CSF, the immature DCs werewashed and subsequently stained with PE anti-human CD86 in combinationwith FITC anti-human CD83. Immature DCs that subsequently had beenactivated for 18 hours with activating factors were also stained with PEanti-human CD86 in combination with FITC anti-human CD83. Mouse IgG1 andIgG2 stained with FITC and PE were used as isotype controls (all from BDBiosciences, California, USA). The samples were analyzed by flowcytometry (FACS) using Cell Quest software (BD Bioscience, California,USA).

Results

Below, the results from the experimental part are commented.

DCs Derived from Co-Cultures of Monocyte-Enriched Allogeneic Leukocytesare not Phenotypically Activated/Mature when Co-Cultured in Aqueous CellCulture Medium Free from Non-Human Serum and Supplemented with GM-CSFand IL-4

Propagation of monocytes from single blood donors in cell culture mediumfree from non-human serum and supplemented with GM-CSF and IL-4 for 4-7days give rise to non-exhausted DCs with a typical “immature” phenotype,including low expression of the maturation marker CD83 and lowexpression of the costimulatory molecule CD86. As seen in FIG. 1 a andb, the mean-expression of both CD83 and CD86 for 3 different “single”DCs was similar as compared to CD83 (FIG. 1 a) and CD86 (FIG. 1 b)expression of DCs derived from a mixture of all three donors. As seen inFIGS. 1 c and d, the strongly increased mean-expression of theactivation/maturation markers CD83 and CD86 for “single” DCs (DCs from 3different peripheral blood donors analysed) after propagation in aqueouscell culture medium free from non-human serum and supplemented withGM-CSF and IL-4 for 4 days and subsequent persistent activation withstimulating factors for 18 hours was similar as compared to CD83 (FIG. 1c) and CD86 (FIG. 1 d) expression on activated DCs derived from amixture of allogeneic monocyte-enriched leukocytes from all threedonors.

Taken together, these findings indicate that monocyte-derived DCs fromthe mixed allogeneic monocyte population are immature after culture inGM-CSF and IL-4 for 5 days and have therefore not experienced anyactivation/maturation signals during their differentiation frommonocytes into immature DCs. Moreover, immature DCs from the mixedallogeneic monocyte-population are at least phenotypically non-exhaustedas they strongly respond with phenotypic maturation when stimulated withactivating factors.

Data obtained with flow cytometry. The respective Y-axis shows the meanfluoredscence intensity (MFI) for CD83 and CD86 before and afterpersistent stimulation with activating factors for 18 hours. The X-axisshow the different combinations measured.

Immature DCs Derived from Co-Cultures of Mixed Allogeneic PeripheralBlood Monocytes are not Functionally Exhausted.

Propagation of monocytes (from one single blood donor) in culture mediumsupplemented GM-CSF and IL-4 for 4-7 days is known to give rise tonon-exhausted DCs which respond with a vigorous production ofpro-inflammatory chemokines (MIP-1 alpha, MIP-1 beta, RANTES and MIG)and pro-inflammatory cytokines (IL-12p70 and TNF-alpha) upon stimulationwith certain activating factors.

As seen in FIG. 2, the high mean levels of MIP-1 alpha (FIG. 2 a), MIP-1beta (FIG. 2 b), RANTES (FIG. 2 c), MIG (FIG. 2 d) produced by “single”DCs (DCs from three different peripheral blood donors analysed) duringpersistent activation with stimulating factors for 18 hours was similaras compared to DCs derived from a mixture of monocytes from all threedonors. Notably, there is a substantial variation in activation-inducedchemokine production between different single donor DCs. As seen in FIG.4, the high mean levels of IL-12p70 (FIG. 3 a) and TNF-alpha (FIG. 3 b)produced by activated “single” DCs was similar as compared to DCsderived from a mixture of monocytes from of all three donors. Notably,there is a substantial variation in IL-12p70 and TNF-alpha productionbetween different single-donor DCs

Data were obtained from ELISA analysis. Results shown are mean values±SDfrom three individuals and the value obtained from the mixture of allthree donors. The respective Y-axis shows the amount of the respectivesubstance produced in pg/mL/1×10⁶ cells, during 18 hours of persistentstimulation/activation. The X-axis show the different combinationsmeasured.

Prostaglandin E2 (PGE2) has been suggested to play a central role inactivation-induced exhaustion of immature DCs (Rieser C et al.,Differential Deactivation of Human Dendritic Cells by EndotoxinDesensitization: Role of Tumor Necrosis Factor-α and Prostaglandin E2.Blood 91 (1998) 3112-3117). We therefore investigated if addition of theCox-2 inhibitor NS-398 (aimed to inhibit potential production of PGE2)during cocultivation of allogeneic monocytes would increase theproduction of proinflammatory chemokines (represented by MIG-production)or proinflammatory cytokines (represented by IL-12p70 production) uponsubsequent activation. As seen in FIG. 4, the presecne of the Cox-2inhibitor NS-398 during propagation of monocytes into DCs did notincrease, but rather decreased, the activation-induced production of MIGand IL-12p70. Thus, there are no signs of PGE2-mediated exhaustion ofdifferentiated immature DCs from cocultures of mixed allogeneicmonocytes.

Data were obtained from ELISA analysis. Results shown are from oneexpreiment from the mixture of all three donors. The respective Y-axisshows the amount of the respective substance produced in pg/mL/1×10⁶cells, during 18 hours of persistent stimulation/activation. The X-axisshow the different combinations measured

Immature DCs Derived from Co-Cultures of Mixed AllogeneicMonocyte-Enriched Buffy Coat Leukocytes are not Functionally Exahusted.

As seen in FIG. 5, the high mean levels of the activation-inducedpro-inflammatory chemokines MIP-1 alpha (FIG. 5 a), MIP-1 beta (FIG. 5b), RANTES (FIG. 5 c), MIG (FIG. 5 d) produced by “single” DCs (DCs fromthree different buffy coat donors analysed) during persistent activationwith stimulating factors for 18 hours was similar as compared to DCsderived from a mixture of monocytes from all three donors. Notably,there is a substantial variation in chemokine production betweendifferent single donor DCs.

As seen in FIG. 6, the high, activation-induced, mean levels of IL-12p70(FIG. 6 a) and TNF-alpha (FIG. 6 b) produced by “single” DCs was similaras compared to DCs derived from a monocyte-enriched leukocyte mixture ofall three donors. Notably, there is a substantial variation in IL-12p70and TNF-alpha production between different single-donor DCs.

Data were obtained from ELISA analysis. Results shown are mean values±SDfrom three individuals and the value obtained from the mixture of allthree donors. The respective Y-axis shows the amount of the respectivesubstance produced in pg/mL/1×10⁶ cells, during 18 hours of persistentstimulation/activation. The X-axis show the different combinationsmeasured.

PI-DCs Derived from Co-Cultures of Mixed Allogeneic Peripheral BloodMonocytes Exhibit a Sustained Production of Pro-Inflammatory Chemokinesand Cytokines

In order to inject activated pro-inflammatory DCs (PI-DCs) intopatients, they usually have to be washed prior to administration. Ifnot, unwanted side-effect induced by the concurrent administration ofstimulating agents (aimed to induce PI-DCs ex vivo) may occur. ImmatureDCs must therefore be activated into PI-DC with sustained production ofdesirable factors also after cessation of the activation-inducingfactors. As seen in FIG. 7, the mean levels of MIP-1 alpha (FIG. 7 a),MIP-1 beta (FIG. 7 b), RANTES (FIG. 7 c), MIG (FIG. 7 d) produced by“single” PI-DCs after withdrawal of activating factors (PI-DCs fromperipheral blood monocytes from three different donors analysed) wassimilar as compared to PI-DCs derived from a mixture of monocytes fromall three peripheral blood donors. Notably, there is a substantialvariation in chemokine production between different single donor PI-DCsafter withdrawal of activation factors. The mean production of IL-12p70(FIG. 8 a) and TNF-alpha (FIG. 8 b) produced by “single” PI-DCs afterwithdrawal of activating factors was also similar as compared to washedPI-DCs derived from a mixture of monocytes from of all three donors.Notably, there is a substantial variation in cytokine production betweendifferent single donor PI-DCs after withdrawal of activation factors.

Data were obtained from ELISA analysis. Results shown are mean values±SDfrom three individuals and the value obtained from the mixture of allthree donors. The respective Y-axis shows the amount of the respectivesubstance produced in pg/mL/1×10⁶ cells during 24 hours after withdrawalof activating factors. The X-axis show the different combinationsmeasured.

PI-DCs Derived from Co-Cultures of Mixed Allogeneic Monocyte-EnrichedPeripheral Buffy Coat Leukocytes Exhibit a Sustained Strong Productionof Pro-Inflammatory Chemokines and Cytokines

As seen in FIG. 9, the mean level of MIP-1 alpha (FIG. 9 a), MIP-1 beta(FIG. 9 b), RANTES (FIG. 9 c), MIG (FIG. 9 d) produced by “single”PI-DCs after withdrawal of activating factors (PI-DCs from buffy coatmonocytes from three different donors analyzed) was similar as comparedto PI-DCs derived from a mixture of monocytes from all three buffy coatdonors. Notably, there is a substantial variation in chemokineproduction between different single donor PI-DCs. The mean production ofIL-12p70 (FIG. 10 a) and TNF-alpha (FIG. 10 b) produced by “single”PI-DCs after withdrawal of activating factors was also similar ascompared to washed PI-DCs derived from a mixture of monocytes from ofall three buffy coat donors. Notably, there is a substantial variationin cytokine production between different single donor PI-DCs.

Data were obtained from ELISA analysis. Results shown are mean values±SDfrom three individuals and the value obtained from the mixture of allthree donors. The respective Y-axis shows the amount of the respectivesubstance produced in pg/mL/1×10⁶ cells during 24 hours after withdrawalof activating factors. The X-axis show the different combinationsmeasured.

Mixed Immature DCs Derived from Monocyte-Enriched Filter LeukocytesProduce Substantial Amounts of Pro-Inflammatory Chemokines and CytokinesUpon Ativation

As seen in FIG. 11, activated mixed DCs derived from monocyte-enrichedfilter leukocytes (the initial leukocyte population was eluted from a4-buffy coat leukocyte depletion filter) produced substantial amountsMIP-1 alpha (FIG. 11 a), MIP-1 beta (FIG. 11 b), RANTES (FIG. 11 c), MIG(FIG. 11 d). As seen in FIG. 12, a substantial amount of IL-12p70 (FIG.12 a) and TNF-alpha (FIG. 12 b) was also produced.

Data were obtained from ELISA analysis. Results shown are values fromone experiment. The respective Y-axis shows the amount of the respectivesubstance produced in pg/mL/1×10⁶ cells, during 18 hours of persistentstimulation/activation

Mixed PI-DCs Derived from Monocyte-Enriched Filter Leukocytes Exhibit aSubstantial Production of Pro-Inflammatory Chemokines and Cytokinesafter Withdrawal of Activating Factors

As seen in FIG. 13, activated mixed DCs derived from filter monocytes(the initial leukocyte population was eluted from a 4-buffy coatleukocyte depletion filter) produced substantial amounts MIP-1 alpha(FIG. 13 a), MIP-1 beta (FIG. 13 b), RANTES (FIG. 13 c), MIG (FIG. 13 d)after withdrawal of activating factors. As seen in FIG. 14, asubstantial amount of IL-12p70 (FIG. 14 a) and TNF-alpha (FIG. 14 b) wasalso produced. Data were obtained from ELISA analysis. Results shown arevalues from one experiment. The respective Y-axis shows the amount ofthe respective substance produced in pg/mL/1×10⁶ cells during 24 hoursafter withdrawal of activating factors.

1. A method of producing non-exhausted immature dendritic cells (DCs),comprising the steps of: providing a mixture of allogeneic leukocytes,which allogeneic leukocytes have been obtained from at least twodifferent, allogeneic donors; isolating allogeneic monocytes from saidmixture of allogeneic leukocytes to provide monocyte-enriched allogeneicleukocytes; and generating non-exhausted immature DCs from saidmonocyte-enriched allogeneic leukocytes, wherein the generation ofnon-exhausted immature dendritic cells (DCs) is performed byco-culturing said monocyte-enriched allogeneic leukocytes for 2 to 7days in aqueous cell culture medium free from non-human serum, saidmedium being supplemented with interleukin-4 (IL-4) andgranulocyte-macrophage colony stimulating factor (GM-CSF).
 2. The methodaccording to claim 1, wherein said cell culture medium comprises atleast one human polypeptide.
 3. The method according to claim 2, whereinsaid human polypeptide is selected from the group consisting oftransferrin, albumin, and insulin.
 4. The method according to claim 1,wherein said monocyte-enriched allogeneic leukocytes comprise allogeneicneutrophils.
 5. The method according to claim 1, wherein said mixture ofallogeneic leukocytes is provided by pooling of at least two buffy coatscomprising leukocytes, said buffy coats to be pooled being obtained fromat least two different, allogeneic donors.
 6. The method according toclaim 5, wherein said pooled buffy coats contain platelets or areplatelet depleted.
 7. The method according to claim 1, wherein saidmixture of allogeneic leukocytes is provided by: eluting leukocytes fromat least two leukocyte depletion filters, which filters, respectively,previously have been used to deplete leukocytes from whole blood, saidwhole blood being obtained from at least two different allogeneicdonors; and pooling the obtained leukocytes to obtain said mixture ofallogeneic leukocytes; or by eluting leukocytes from a leukocytedepletion filter, which filter has been used to deplete leukocytes frompooled buffy coats, wherein the pooled buffy coats originate from atleast two different, allogeneic donors.
 8. The method according to claim1, wherein said allogeneic monocytes are isolated by elutriation or byantibody/bead isolation.
 9. The method according to claim 1, whereinsaid co-culturing is performed for about 5 days.
 10. The methodaccording to claim 1, further comprising the step of loading thenon-exhausted immature DCs with an antigen.
 11. (canceled)
 12. A methodof producing pro-inflammatory DCs, comprising the steps of: providingnon-exhausted immature DCs according to claim 1; and activating thenon-exhausted immature DCs to obtain pro-inflammatory DCs.
 13. Themethod according to claim 12, wherein said activation is performed byaddition of the Toll-like receptor 3 (TLR3)-ligand poly-I:C, aTLR7/8-ligand, and interferon gamma (IFN-γ) to induce activation. 14.The method according to claim 13, wherein said activation furthercomprises the addition of at least one substance selected from the groupconsisting of TLR2-ligands, TLR4-ligands, TLR9-ligands, Interferon alpha(IFN-α), interleukin 1β (IL-1β), and tumor necrosis factor alpha (TNF-α)to induce activation.
 15. The method according to claim 13, wherein saidactivation does not comprise addition of prostaglandin E2 (PGE2). 16.The method according to claim 13, wherein the immature DCs are exposedto the activation factors for 8 to 24 hours, whereafter essentially allof the activation factors are washed away.
 17. A mixture of allogeneicpro-inflammatory dendritic cells originating from at least twodifferent, allogeneic donors, said dendritic cells being obtainable by amethod according to claim
 12. 18. A pharmaceutical compositioncomprising the mixture of allogeneic pro-inflammatory dendritic cellsaccording to claim 17 and at least one pharmaceutical acceptablecarrier.
 19. A method of treating cancer said method comprisingadministering the composition according to claim 18 to a subject in needthereof, wherein said composition comprises a therapeutically effectiveamount of a mixture of allogeneic pro-inflammatory dendritic cellsaccording to claim
 17. 20. The method according to claim 13, whereinsaid TLR7/8-ligand is selected from the group consisting of Resiquimod,Gardiquimod and Imiquimod.